BQ-123

Endothelin (ET) is a peptide comprising 21 amino acids, and its G-protein coupled ETA and ETB receptors are expressed in both cancer cells and cancer-associated cells. Cancer cells excessively express ETA and ETB receptors, and mechanical stimulation promotes the growth and migration of these cells. The autocrine and paracrine signaling of ET is involved in several cancer metabolic pathways, but how this is mediated by the ET-axis (ET and its receptors) remains to be clarified. This study investigated ET-axis-mediated autocrine/paracrine communication in rat squamous cell carcinoma (SCC) in response to direct mechanical stimulation of such cells. Intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was determined using fura-2/AM from a rat SCC cell line (SCC-158). Direct mechanical stimulation of the SCC-158 cells using glass micropipettes to compress the cell membrane to 8 μm for 4 sec induced a transient increase in [Ca²⁺]_i. This increase was also observed in the neighboring cells of the stimulated SCC-158 cells. Treatment with 10 μM Gd³⁺ or 1 μM GsMTx4 almost completely inhibited the mechanical stimulation-induced increase in [Ca²⁺]_i in the SCC-158 cells. Application of 1 μM BQ-123 (an ETA receptor antagonist) inhibited the increase in [Ca²⁺]_i in the stimulated SCC-158 and neighboring cells, whereas that of BQ-788 (an ETB receptor antagonist) had no such effect. These findings suggest that rat SCC cells express the Piezo1 channel. Activation of the Piezo1 channel induced endothelin release from the mechanically stimulated SCC-158 cells. Released ET activated ETA receptors in neighboring SCC-158 cells. These findings also suggest that intercellular paracrine communication among SCC-158 cells through ET signaling plays an important role in the development and metabolism of SCC cells.

To study the role of perivascular adipose tissue (PVAT) in the reactivity of rat and human vessels.

Iliac and mesenteric arteries were obtained from normotensive Sprague-Dawley rats, hypertensive transgenic (mRen2)27 rats overexpressing mouse renin, and (mRen2)27 rats made diabetic with streptozotocin. Human coronary arteries were obtained from donors. Concentration-response curves were constructed to endothelin-1 and acetylcholine with and without PVAT. The contribution of NO and endothelium-dependent hyperpolarization (EDH) were determined making use of the NO synthase inhibitor L-NAME and the EDH inhibitors apamin + TRAM-34. The endothelin type A and type B (ET_A, ET_B) receptor blockers BQ123 and BQ788, the chemerin inhibitors α-NETA and pravastatin, and the angiotensin receptor blocker losartan were also used.

In rat iliac arteries, PVAT diminished endothelin-induced constriction, while the opposite was true in human coronaries. Coronary effects were unaltered by α-NETA, pravastatin, or losartan. ET_B receptor-mediated relaxation in iliac arteries occurred only with PVAT, and BQ123 blocked endothelin-1-induced constriction. Diabetes upregulated the anticontractile effects of PVAT. In rat mesenteric arteries, acetylcholine-induced relaxation with PVAT relied on NO, and on NO + EDH without PVAT. Diabetes upregulated the EDH component exclusively with PVAT.

PVAT modulates ET-1-induced constriction in a vessel type-dependent manner. Its enhancing effects in coronaries involved neither chemerin nor angiotensin II. Its anticontractile effects in rat iliac arteries involved ET_B receptor-mediated relaxation. Diabetes upregulated PVAT's anticontractile effects. In mesenteric arteries, PVAT counterbalanced the EDH component of the relaxant effect of acetylcholine. Diabetes reversed this effect by upregulating the EDH component.